Phenotypic characterization and in situ genotyping of a library of genetically different cells

ABSTRACT

A library of cell strains is characterized by culturing cells at spatially defined and separated positions in a culturing device ( 1 ) and determining a phenotypic characteristic of each cell strain in the culturing device ( 1 ). The cells are fixated at the spatially defined and separated positions in the culturing device ( 1 ) followed by in situgenotyping a respective variable region ( 150 ) of each cell strain at the spatially defined and separated positions in the culturing device ( 1 ). Each respective phenotypic characteristic is connected to each respective genotype based on the spatially defined and separated positions in the culturing device ( 1 ).

TECHNICAL FIELD

The present embodiments generally relate to phenotypic characterizationand in situ genotyping of a library of genetically different cells.

BACKGROUND

The recent development in genome engineering, for instance exemplifiedby Multiplex Automated Genomic Engineering (MAGE) and applications ofClustered Regularly Interspaced Short Palindromic Repeats (CRISPR)associated protein 9 (Cas9) or other types of genome editing facilitatedby engineered nucleases, such as Zinc finger nucleases (ZFNs) andTranscription Activator-Like Effector Nucleases (TALENs), in combinationwith decreased costs for DNA oligonucleotide synthesis, makes itpossible to generate large cell libraries with overwhelming geneticdiversity. At the same time technology development has led to thepossibility of determining phenotypic and genotypic characteristics ofcells. For instance, Fluidigm Dynamic Array™ integrated fluidic circuits(IFC) can be used for genotyping in a single microwell plate. However,the size of the cell libraries that can be analyzed is restricted to afew hundred different cells since the genetically different cells mustbe kept sorted and analyzed individually.

Another technology that can handle significantly larger strain librariesthan Fluidigm Dynamic Array™ IFC is fluorescence-activated cell sorting(FACS). However, FACS has limitations since the phenotypiccharacterization of the cell libraries is limited to fluorescencereadout at a single point in time. In vitro compartmentalization (IVC)and droplet-based technology can, similar to FACS, handle largelibraries. However, the phenotypic information obtained in IVC islimited because of optical constraints for imaging in droplets and bythe fact that long term cell growth experiments cannot be performed inthe droplets.

Yet other techniques depend on adding barcoded oligomers to cells afterimaging. The need for distribution of oligomers to specific spatialpositions limits the conditions under which cells can be grown and thenumber of different barcodes that can be distributed.

Thus, there is a need for improvements within the technical field ofphenotypic and genotypic characterization of cell libraries. Inparticular, there is a need for a technology that can handle large celllibraries and that can monitor phenotypic characteristics with highspatial and temporal resolution.

SUMMARY

It is a general objective to provide a technology that allows phenotypicand genotypic characterization of large cell libraries.

This and other objectives are met by embodiments as defined herein.

Briefly, an aspect of the embodiments relates to a method forcharacterizing a library of a plurality of cell strains having differentvariable regions in at least one part of the genetic material of thecell strains. The method comprises culturing cells of the cell strainsat spatially defined and separated positions in a culturing device. Aphenotypic characteristic is determined of each cell strain in theculturing device. The cells of the cell strains are fixated at thespatially defined and separated positions in the culturing device. Themethod also comprises in situ genotyping the variable region of eachcell strain at the spatially defined and separated positions in theculturing device. Each respective phenotypic characteristic is thenconnected to each respective genotype based on the spatially defined andseparated positions in the culturing device.

Another aspect of the embodiments relates to a system for characterizinga library of a plurality of cell strains having different variableregions in at least one part of the genetic material of the cellstrains. The system comprises a culturing device configured to culturecells of the cell strains at spatially defined and separated positionsin the culturing device. The system also comprises a first kitcomprising components for in situ genotyping the variable region of eachcell strain following fixation of the cells at the spatially defined andseparated positions in the culturing device. A respective phenotypiccharacteristic of each cell strain can then be connected to eachrespective genotype based on the spatially defined and separatedpositions in the culturing device.

The present embodiments enable parallel phenotypic and genotypiccharacterization of large libraries of cells. The embodiments furtherallow various phenotypes to be monitored and determined for the cells inthe culturing device.

The system and culturing device of the embodiments facilitate longsingle cells phenotyping experiments under constant or variableconditions followed by fixation and application of multiple reagentsneeded for in situ sequencing without losing cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments, together with further objects and advantages thereof,may best be understood by making reference to the following descriptiontaken together with the accompanying drawings, in which:

FIG. 1 is an illustration of a culturing device according to anembodiment;

FIG. 2 is a cross-sectional view of the culturing device shown in FIG. 1along the line A-A;

FIG. 3 is an illustration of a culturing device according to anotherembodiment;

FIG. 4 is a cross-sectional view of the culturing device shown in FIG. 3along the line A-A;

FIG. 5 is an illustration of a culturing device according to a furtherembodiment;

FIG. 6 is an illustration of a culturing device according to yet anotherembodiment;

FIG. 7 is an illustration of a culturing device according to a furtherembodiment;

FIG. 8 illustrates part of the genome of a cell strain according to anembodiment;

FIG. 9 illustrates a mRNA sequence obtained from the part of the genomeshown in FIG. 8; and

FIG. 10 illustrates a cDNA sequence obtained from the mRNA sequenceshown in FIG. 9;

FIG. 11 illustrates part of the genome of a cell strain according toanother embodiment; and

FIG. 12 illustrates a cDNA sequence obtained from the part of the genomeshown in FIG. 11.

DETAILED DESCRIPTION

Throughout the drawings, the same reference numbers are used for similaror corresponding elements.

The present embodiments generally relate to phenotypic characterizationand in situ genotyping of a library of cell strains. In particular, thepresent embodiments allow monitored phenotypic characteristics and insitu determined genotypes to be connected in a highly parallel way. Thismeans that a vast library of cell strains with different genotypes canbe processed in parallel in order to connect the monitored phenotypiccharacteristics to the different genotypes of the cell strains.

Cell strain as used herein denotes cells derived from a primary cultureor a cell line by selection and cloning of cells having specificgenotype. Thus, cells of a cell strain all have the same genotype. Alibrary of cell strains is thereby a collection of genetically differentcells. The cell strains in the library of the embodiments can be anycell type including bacterial strains, yeast strains, eukaryotic cellstrains, cell lines, primary cells, stem cells, cells in tissues ormicrocolonies, isogenic cells that are different with respect to mobiledeoxyribonucleic acid (DNA) elements, vectors or plasmids that theycarry. Cell strains as used herein also encompass multicellularcomplexes, tissues, etc. as long as these can be cultured in vitro asdisclosed herein.

An aspect of the embodiments relates to a method for characterizing alibrary of a plurality of cell strains having different variable regionsin at least one part of the genetic material of the cell strains. Themethod comprises culturing cells of the cell strains at spatiallydefined and separated positions in a culturing device. A phenotypiccharacteristic of each cell strain is determined in the culturingdevice. The cells of the cell strains are fixated at the spatiallydefined and separated positions in the culturing device. The method alsocomprises in situ genotyping the variable region of each cell strain atthe spatially defined and separated positions in the culturing device.Each respective phenotypic characteristic is then connected to eachrespective genotype based on the spatially defined and separatedpositions in the culturing device.

The library of cell strains can be obtained according to varioustechniques within genome engineering. For instance, Multiplex AutomatedGenomic Engineering (MAGE) can be used to create several billions ofdifferent mutant genomes per day (Wang et al. Nature, 2009, 460:894-898). Other techniques that can be used to create a library of cellsinclude Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR) associated protein 9 (Cas9) (Wang et al., Science, 2014, 343:80-84; Koike-Yusa et al., Nature Biotechnology, 2014, 32: 309-312; Zhouet al., Nature, 2014, 509: 487-491) or large-scale RNA interference(Berns et al., Nature, 2004, 428: 431-437).

The present embodiments use a culturing device in which cells of eachcell strain in the library can be kept and cultured separately fromcells of other cell strains and or other genotypes. Hence, each cellstrain has a respectively spatially defined and separated position inthe culturing device in which the cells can grow and be studied.

Culture medium or media may advantageously be added to the cells at thespatially defined and separated positions in the culturing device. Theculture medium could be continuously added to the cells or isreplenished or exchanged periodically or at selected time instances. Themedia exchange is preferably in such a way that excess cells do notattach and contaminate the spatially defined and separated positions ofcells of other genotypes.

The cells are preferably allowed to be cultured and grown in a monolayerat the spatially defined and separated positions in the culturingdevice. A monolayer is generally preferred over 3D structures ormatrices in terms of that is typically easier to monitor the cells anddetermine phenotypic characteristics of the cells if they are present ina monolayer. However, cells can be grown in structures that support 3Dgrowth if phenotypes related to, for instance, cell-to-cell interactionsare monitored, such as for instance in differentiation or development.

The cells cultured at the spatially defined and separated positions inthe culturing device can preferably be exposed to various physicaland/or chemical stimuli or agents without being washed away, in order tomonitor the response of the cells to the physical and/or chemicalstimuli or agents. For instance, various chemical test agents, such asnutrients, drugs, antibiotics, gene expression inducers or repressors,could be added to the culture medium and thereby contact the cells. Thephenotypic characteristics of the cell strains in terms of the responseof the cells of the different cell strains to the various test agentscan then be determined, for instance, using microscopy. Correspondingly,the temperature, pH, pressure, flow, gases, light exposure or mechanicalstress that the cells are exposed to could be changed and the responseof the cells of the different cell strains to such changing physicalconditions can be determined, for instance, using microscopy.

The cells strains have different genotypes as represented by havingdifferent variable regions in at least one part of their geneticmaterial. The variable region is typically present in the genome of thecell strains. Alternatively, the variable region is present in a mobilegenetic element, such as plasmid or vector, and hence does notnecessarily have to be stably incorporated into the genome of the cells.In the following, the embodiments are mainly discussed with regard tothe variable region being present in the genome. However, alternativeembodiments are possible where the variable region and the further DNAelements mentioned herein are instead present in a plasmid or othermobile genetic element of the cell strains. The variable region, orparts thereof, can also be present in unstable genetic elements, such astransposons, viruses or phages. Such encoding will sometimes haveadvantages in terms of amplifying the variable sequence before fixingthe cells for in situ sequencing, for example by specifically excisingor circularizing parts of the variable region from the dsDNA genomebefore fixing the cells.

Various culturing devices that can be used according to the embodimentswill be further described herein.

In an embodiment, the method also comprises randomly seeding cells ofthe cell strains at the spatially defined and separated positions in theculturing device. The randomly seeding of cells is preferably performedso that each spatially defined and separated position only comprisescells of a same genotype, i.e. of the same cell strain.

An advantage of the embodiments is that the genetic identity, i.e.genotype, of the cells does not need to be determined and known prior toseeding of the cells in the culturing device. Thus, there is no need tokeep the cells in the cell library sorted prior to seeding in terms ofhaving to know the genetic identity of each cell strain and continuouslymonitoring the position of each genotype throughout the method. Thismeans that the present embodiment in clear contrast first analyzes thephenotypic characteristics in parallel without any knowledge of thegenotype and then determines the genotypes and connects them to thephenotypic characteristics.

By in situ sequencing individual cell strains it is possible to grow thedifferent strains much denser than what would be possible if, forexample, specific barcoded sequencing primers would be distributed tothe spatially separated positions before or after phenotyping.

The random seeding can thereby be performed by letting individual cellsfrom a library generated in bulk settle at spatially defined andseparated positions and form a microcolony of isogenic cells.

In an alternative approach, the random seeding can be performed byadding cells of a first cell strain in the library to a first spatiallydefined and separated position in the culturing device, a second cellstrain in the library to a second spatially defined and separatedposition in the culturing device, and so on until all cell strains to bemonitored have been distributed among the different spatially definedand separated positions in the culturing device.

The phenotypic characteristic determined for each cell strain in thelibrary is preferably a phenotypic characteristic corresponding to eachgenotype in the library. Thus, the cells in the library are geneticallydifferent cells having different genotype, i.e. a respective genotypeper cell strain. The differences in genotype imply that the cells willhave different phenotypic characteristics corresponding to eachrespective genotype.

In an embodiment, the phenotypic characteristics of the cells aredetermined using microscopy. Microscopy for monitoring and determiningphenotypes has several advantages as compared to prior art technologies.For instance, fluorescence microscopy allows for extensive time laps ofcell linages over many generations, single molecule detectionsensitivity and the possibility to monitor temporal responses tochanging growth conditions in any way. Thus, it is possible to monitorthe phenotype of the cell lines in parallel over an extended period oftime and not only at a single time point as in FACS.

Non-limiting but illustrative examples of phenotypic characteristicsthat can be monitored and determined according to the embodiments usingmicroscopy include cell morphology, spatial and/or temporal expressionpatterns of various molecules, such as ribonucleic acid (RNA) orproteins, levels of specific metabolites, lifespan or growth ratechanges, such as in response to addition of different physical orchemical stimuli or agents, cell-to-cell variations in gene expressionlevels, embryo development, brightness of reporter proteins or RNAaptamers, etc.

The phenotypic characterization of the cell strains can, thus, beperformed in parallel under a microscope for a long period of time ifneeded. The phenotypic characterization is furthermore performed withoutknowledge of the genotype of the various cell strains in the library. Inclear contrast, the phenotypic characterization instead determinesrespective phenotypic characteristics for each spatially defined andseparated position in the culturing device. For instance, assume thatthe relevant phenotypic characteristic to be determined for the cellstrains is gene expression of a target gene for a fluorescence reporterprotein, with a variable gene regulatory region or coding sequence,following addition of a test agent. In such a case, the microscope canbe used to take an image over the culturing device in which therespective gene expression levels can be visually determined. Eachindividual gene expression level can then be quantized to get arespective value for each spatially defined and separated position inthe culturing device. Thus, the output of the determination of thephenotypic characterization could be a list or matrix of one or morerespective values for each spatially defined and separated position inthe culturing device.

It is advantageously if the spatially defined positions are labeled,numbered or marked so that it is easy to define the relationship betweenspatially defined position and phenotype, i.e. which spatially definedposition corresponds to which phenotype. It could also be possible todefine the spatial position in relation to the distance or order amongthe spatially defined positions.

For instance, a list could be in the form of position 0=value 0,position 1=value 1, position 2=value 2, and so on. A matrix maycorrespondingly be in the form of position (x, y)=value 0, position(x+1, y)=value 1, and so on.

In an embodiment, determining the phenotypic characteristic comprisesdetermining the phenotypic characteristic of each cell strain duringculturing of the cells in the culturing device using microscopy.Examples of microscopy technologies that can be used in the embodimentsinclude, for instance, bright field microscopy, phase contrastmicroscopy, fluorescence microscopy, light sheet microscopy, or any typeof super resolution imaging modality such as stimulated emissiondepletion (STED) microscopy, photo-activated localization microscopy(PALM), near-field scanning optical microscopy (NSOM), 4Pi microscopy,structured illumination microscopy (SIM), ground state depletion (GSD)microscopy, spectral precision distance microscopy (SPDM), stochasticoptical reconstruction microscopy (STORM). Furthermore, IntracellularSingle Particle Tracking (SPT) or Fluorescence Correlation Spectroscopy(FCS) could also be used. Microscopy analysis can be made at fixed timepoints or using time lapse imaging.

Other measurements of the phenotypes are also possible, such asmeasuring mechanical properties using atomic force microscopy, membranepotential using indicator dyes or micro electrodes, small moleculesecretion using imaging mass spectrometry or specified biosensor arrays.Near-field optical array detectors directly connected to the culturingdevice are also possible.

Once the phenotypic characteristics of the cell strains have beendetermined the cells are preferably fixated at the spatially defined andseparated positions in the culturing device. Cell fixation can beperformed according to techniques well known in the art. For instance,formaldehyde can be used for cell fixation. In a non-limiting examplecells are fixed with 4% formaldehyde for about 15 minutes or 3% (w/v)paraformaldehyde in phosphate buffered saline (PBS) for about 30minutes.

In an embodiment, the fixated cells are permeabilized prior to in situgenotyping. Various protocols traditionally employed for cellpermeabilization can be used according to the embodiments. For instance,Triton X-100 (such as 0.25% Triton X-100) or another surfactant, such asnonionic surfactant, can be used. Alternatively, ethanol, such as 70%ethanol, can be used for cell permeabilization. Further examples includehydrochloric acid, such as 0.1 M hydrochloric acid, optionally combinedwith a protease, such as pepsin, e.g. 0.01% pepsin, or lysozyme todegrade the bacterial cell wall.

In an embodiment, the cells are induced to express or activate one ormore enzymes to modify or amplify the variable sequence or parts thereofbefore fixation. Non limiting examples include activation ordeactivation of transcription factors or RNA polymerases that lead totranscription of a barcode and neighboring sequences into RNA,restriction enzymes that cut the variable sequence from chromosomal DNA,transposases that excide DNA including the variable sequence, ligasesthat ligates ssDNA or dsDNA to form templates for rolling circleamplification, etc.

The in situ genotyping comprises in situ genotyping at least a part ofthe variable region of each cell strain at the spatially defined andseparated positions in the culturing device. Hence, it is not absolutelynecessary to in situ genotype the complete variable region of each cellstrain. Hence, in situ sequencing as used herein comprises in situsequencing at least a part of the variable region or indeed the completevariable region. The in situ sequencing preferably outputs informationshowing any nucleotide differences in the variable region betweendifferent cell strains and where these nucleotide differences give riseto different phenotypes.

In an embodiment, in situ genotyping is based on the technologyfluorescent in situ sequencing (FISSEQ) as described for example inScience, 2014, 343(6177): 1360-1363. Briefly, in FISSEQ cDNA ampliconswithin the cell are generated in the fixed cells using reversetranscriptase and incorporation of aminoallyl deoxyuridine5′-triphosphate (dUTP) during reverse transcription (RT). The cDNA isrefixed using BS(PEG) 9, an amine-reactive linker with a 4 nm spacer.The cDNA fragments are then circularized before rolling circleamplification (RCA). BA(PEG)9 is then used to cross-link the RCAamplicons containing aminoallyl dUTP. SOLiD sequencing by ligation canthen be used to sequence the relevant sequence in the RCA amplicons toget the nucleotide sequence of the variable region.

In an embodiment, in situ genotyping the variable region preferablycomprises in situ sequencing by ligation of the variable region or atleast a portion thereof at the spatially defined and separated positionsin the culturing device. Sequencing by ligation relies upon thesensitivity of deoxyribonucleic acid (DNA) ligase for base pairmismatches. Generally, the variable region to be sequenced is preferablyin the form of a single stranded DNA sequence, flanked on at least oneend by a known sequence that will function as anchor primer-bindingsequence. An anchor primer that is complementary to the known sequenceis brought to bind to the known sequence.

A mixed pool of probe oligonucleotides, typically eight to nine baseslong, is then brought in, labelled, typically with a fluorescent dye,according to the position that will be sequenced. These labelledoligonucleotides hybridize to the variable region, next to the anchorprimer and DNA ligase preferentially joins an oligonucleotide to theanchor primer when its nucleotide sequence matches the unknown variableregion. Based on the fluorescence produced by the molecule, one caninfer the identity of the base at this position of the variable region.

The oligonucleotide probes may also be constructed with cleavablelinkages, which can be cleaved after identifying the label. This willboth remove the label and regenerate a 5′-phosphate on the end of theligated probe, thereby enabling a new round of ligation. This cycle ofligation and cleavage can be repeated several times to read longersequences. This technique sequences every N^(th) base in the variableregion, where N is the length of the probe left behind after cleavage.In order to sequence the skipped positions in the variable region, theanchor primer and the ligated oligonucleotides may be stripped of thevariable region, and another round of sequencing by ligation is startedwith an anchor primer that is one or more bases shorter.

Another technique is to do repeated rounds of a single ligation wherethe label corresponds to different positions in the probe, followed bystripping the anchor primer and ligated probe.

Sequencing by ligation can be proceeded in either direction (5′-3′ or3′-5′) depending on which end of the oligonucleotide probes that isblocked by the label.

In an embodiment, the sequence that is sequenced is preferably acomplementary DNA (cDNA) sequence obtained by reverse transcription ofan RNA transcript obtained from the variable region. In this embodiment,the variable region is flanked by at least one known sequence to whichthe anchor primer will bind.

Sequencing by ligation can be performed on fixated cells to achieve anin situ sequencing by ligation of the variable region or at least aportion thereof at the spatially defined and separated positions in theculturing device, see for instance Science 2014, 343: 1360-1363 andNature Methods 2013, 10: 857-860, the teachings of which are herebyincorporated by reference with regard to performing in situ sequencingby ligation.

Briefly, in one variant, RNA obtained from the variable region, or abarcode see further below, is copied to cDNA by reverse transcription,followed by degradation of the mRNA strand using an RNase.

In a first embodiment, a padlock probe binds to the cDNA with a gapbetween the probe ends over the bases that are targeted for sequencingby ligation. This gap is filled by DNA polymerization and DNA ligationto create a DNA circle.

In a second embodiment, cDNA circulation is carried out by ssDNAligation only.

In a third embodiment, dsDNA including at least a part of the variablesequence and neighboring DNA is excised from the surrounding DNA, by forexample restriction enzymes or transposases. The excised dsDNA can thenbe digested to ssDNA by endonucleases in order to self-hybridize andligate to form a circular DNA.

In either case, the formed DNA circle is amplified by target-primedrolling circle amplification (RCA) generating a rolling circle product(RCP) that is subjected to sequencing by ligation. An anchor primer ishybridized next to the targeted sequence before the ligation ofoligonucleotides probes. In an embodiment, the oligonucleotide probesconsist of four libraries of 9-mers, with eight random positions (N) andone fixed position (A, C, G or T). Each library is labeled with one offour fluorescent dyes. The oligonucleotide probe with best match at thefixed position will be incorporated by ligation along with itsfluorescent label. The sample is imaged and each RCP displays the colorcorresponding to the matched base. The oligonucleotide probe is washedaway before the application of oligonucleotide probes for the next base.The steps of ligation, washing, imaging and stripping are iterated untilthe desired number of bases has been read.

In another embodiment, in situ genotyping comprises in situ sequencingby synthesis of the variable region or at least a portion thereof at thespatially defined and separated positions in the culturing device.

For instance, four types of modified dNTPs containing a terminator thatblocks further polymerization are added. The terminator also contains afluorescent label that can be detected by camera. Non-incorporatednucleotides are washed away and images of the fluorescently labelednucleotides are taken. The fluorescent label along with the terminatorare chemically removed from the DNA allowing for the next cycle ofsequencing to being.

The result of the in situ genotyping is preferably the nucleotidesequence of the variable region or at least a portion thereof for eachcell strain. Each nucleotide sequence is furthermore connected to arespective spatially defined and separated position in the culturingdevice. This is possible since the genotyping is performed as an in situgenotyping, such as in situ sequencing by ligation or synthesis. In situhere implies that the genotyping is performed on site or in position,i.e. in the spatially defined and separated positions in the culturingdevice.

The output of the previously described phenotyping was a respectivedetermined phenotypic characteristic for each spatially defined andseparated position in the culturing device, such as in the form of alist or matrix listing the phenotypic characteristic(s) determined foreach spatially defined and separated position.

The output of the in situ genotyping is the nucleotide sequencedetermined for the variable regions at each spatially defined andseparated position in the culturing device. This output may also be inthe form of a list or matrix listing the nucleotide sequence determinedfor each spatially defined and separated position.

For instance, a list could be in the form of position 0=sequence 0,position 1=sequence 1, position 2=sequence 2, and so on. A matrix may bein the form of position (x, y)=sequence 0, position (x+1, y)=sequence 1,and so on.

Each respective phenotypic characteristic can then be connected orassociated with each respective genotype based on the spatially definedand separated positions in the culturing device. For instance, thephenotypic characteristic determined for the cells of the cell strain atposition 0 in the culturing device is a result of the genotype of thecells of this cell strain and this genotype is obtained from thenucleotide sequence determined for position 0. Hence, the connection ofphenotype and genotype can be achieved simply by matching the phenotypiccharacteristics and genotypes determined for each spatially defined andseparated position in the culturing device.

In an embodiment, each cell strain has a respective strain-specificbarcode sequence 140 in its genetic material, preferably in its genome100, see FIG. 8. In such a case, the in situ genotyping preferablycomprises determining the respective genotype by in situ sequencing atleast a part of the respective barcode sequence 140 of each cell strainat the spatially defined and separated positions in the culturing device1.

Thus, the barcode sequence 140 provides a bridge to the determination ofthe variable region 150 in a cell strain. In particular, the in situgenotyping can be performed by in situ sequencing, such as in situsequencing by ligation or synthesis, the comparatively much shorterbarcode sequence 140 instead of sequencing the variable region 150. Thenucleotide sequence of the variable region 150 can then be obtainedbased on the sequenced barcode sequence 140 and mapping information asdescribed below.

The method preferably comprises determining mapping informationspecifying a connection between each variable region 150 and arespective barcode sequence 140. Determining the respective genotypethen preferably comprises determining the respective genotype based onthe in situ sequenced at least a part of the respective barcode sequenceand the mapping information.

The mapping information can thereby be regarded as a look-up table thatoutputs the nucleotide sequence of a variable region 150 given an inputnucleotide sequence of at least a part of a barcode sequence 140. Themapping information could thereby be in the form of a table listing therespective barcode sequence 140 for each variable region 150.

The mapping information can be obtained in connection with producing thelibrary of cell strains. For instance, genome engineering can be used togenerate large libraries of cell strains with genetic diversity withregard to the variable region 150 as represented by various pointmutations 155 in FIG. 8. Such technology can also be used to tailorrespective barcode sequences 140 for the different variable regions 150.For instance, the library of cell strains could be designed so thatbarcode sequence no. 1 is included in the genome 100 of the cell strainhaving variable sequence no. 1, and so forth. This typically requiresthat the barcode 140 is embedded in the variable region 150 or in itsimmediate proximity.

In another embodiment, the barcode sequence 140 and the variable region150 are not introduced at the same reaction. The mapping information maythen be determined by sequencing the library of cell strains in bulk toobtain, for each cell strain, a sequence read encompassing the variableregion 150 and the respective specific barcode sequence 140. This allowsthe barcode sequence 140 to be as far away from the variable region 150as is it is possible to make in a single sequencing read.

Thus, the relevant region of the genome or chromosome of most cells inthe library is sequenced in bulk in such a way that individual readsencompass the variable region 150 and the barcode sequence 140. Theresult of the bulk sequencing is thereby the nucleotide sequence of thesequence read for each cell strain. This information can thereby bestored and used as mapping information.

The barcode sequence 140 can also be introduced in another position in achromosome or be maintained in a mobile genetic element, such as aplasmid. In these cases the barcode sequence 140 is connected to thevariable region 150 by the method of introducing the variable region 150and barcode sequence 140 into the cell at the same time. For example,the variable region 150 can be delivered to the cells as a part of aplasmid that also carries the corresponding barcode sequence 140.

In an embodiment, each cell strain has a construct comprising arespective strain-specific barcode sequence 140 and the variable region150 in its genome 100 flanked by library-common primer-binding sequences160, 170 of know nucleotide sequence. The library-common primer-bindingsequences 160, 170 can be used to amplify the respective strain-specificbarcode sequence 140. Alternatively, the at least one library-commonprimer-binding sequence 160, 170 can be used to sequence the respectivestrain-specific barcode sequence 140 directly from the genome 100 or viaa transcribed RNA sequence 200, see FIG. 9, that is reverse transcribedinto a cDNA sequence 300, see FIG. 10.

Hence, in this embodiment each cell strain has a barcode sequence 140 inits genome 100 or a mobile genetic element. This barcode sequence 140 isadvantageously created together with the variable region 150 whencreating the library of cell strains as previously described herein. Thebarcode sequence 140 is strain-specific implying that each cell strain,i.e. genotype or version of the variable region 150, has its ownspecific nucleotide sequence for the barcode sequence 140. Thus, thelibrary preferably does not contain two different cell strains withdifferent variable regions 150 but having the same barcode sequence 140.

In contrast to the strain-specific barcode sequence 140, the at leastone primer-binding sequence 160, 170 is preferably library-common orstrain-common implying that this at least one primer-binding sequence160, 170 preferably has the same nucleotide sequence in all cell strainsof the library. Hence, one and the same primer or primer pair that iscomplementary to the primer-binding sequence 160 or sequences 160, 170can thereby be used for amplification or sequencing purposes in all cellstrains.

In a first embodiment, the construct comprises one library-commonprimer-binding sequence 160, 170. In such a case, the library-commonprimer-binding sequence 160 could be provided upstream of the barcodesequence 140 (and the variable region 150) or the library-commonprimer-binding sequence 170 is downstream of the barcode sequence 140(and the variable region 150).

In a second embodiment, the construct comprises two library-commonprimer-binding sequences 160, 170. In such a case, one is preferablyprovided upstream of the barcode sequence 140 (and the variable region150) and the other is positioned downstream of the barcode sequence 140(and the variable region 150) as shown in FIG. 8. These twolibrary-common primer-binding sequences 160, 170 can then be used asbinding sites for a padlock probe around the barcode sequence 140.

FIG. 8 illustrates an example of a portion of the genome 100 in a cellstrain of the library. FIG. 9 represents a transcribed RNA sequence 200or mRNA sequence 200 obtained by transcribing the construct with thestrain-specific barcode sequence 140, the variable region 150 and the atleast one library-common primer-binding sequence 160, 170. FIG. 10represents a cDNA sequence 300 obtained by reverse transcribing the RNAsequence 200 of FIG. 9.

In FIGS. 8-10 reference numbers 1X0, 2X0, 3X0 have been used, with X=3-7to represent the corresponding nucleotide sequence portions in thegenome 100, in the transcribed RNA sequence 200 or in the cDNA sequence300.

FIG. 8 additionally shows a promoter sequence 110 used to transcribe theconstruct comprising the strain-specific barcode sequence 140, thevariable region 150 and the at least one library-common primer-bindingsequence 160, 170. The genome 100 may optionally comprise at least oneregulatory sequence 120, which controls the promoter sequence 110 andtranscription of the construct.

In an embodiment, the library-common primer-binding sequences 160, 170are used to amplify the strain-specific barcode sequence 140 andoptionally also the variable region 150, for instance by in situpolymerase chain reaction (PCR). The amplification of at least thestrain-specific barcode sequence 140 may be advantageous prior to insitu sequencing the strain-specific barcode sequence 140 in order to geta sufficient copy number of the strain-specific barcode sequence 140 toperform the in situ sequencing.

The amplification can be performed directly on the genome sequence 100in the cell libraries. However, it may be preferred to first perform insitu reverse transcription of the transcribed RNA sequence 200 obtainedby transcribing the construct with the strain-specific barcode sequence140, possibly the variable region 150 and the at least onelibrary-common primer-binding sequence 160, 170 in the genome 100. Insuch a case, a DNA primer 360 complementary to a library-commonprimer-binding sequence 260 in the RNA sequence 200 is added togetherwith a reverse transcriptase or an RNA-dependent DNA polymerase to insitu generate the cDNA sequence 300 as schematically illustrated in FIG.9. The amplification is then made of the generated cDNA sequence 300.

The amplification may, in an embodiment, be in the form of in situamplification of the cDNA sequences 300 by rolling circle amplification(RCA) following circulation using single-stranded DNA (ssDNA) ligase orhybridization and ligation of a padlock probe. Alternatively, the insitu amplification of the cDNA sequences 300 can be performed by in situPCR.

Usage of a padlock probe in order to form a circular DNA sequence from acDNA sequence is described in Nature Methods 2013, 10: 857-860, see forinstance FIG. 1 in that document.

The resulting cDNA sequence 300 can then be amplified by means of a DNApolymerase and a DNA primer complementary to a library-commonprimer-binding sequence 360 in the cDNA sequence 300. In situ sequencingof the strain-specific barcode sequence 360 in the resulting cDNAsequences 300 can then take place as further described herein.

In an embodiment, the method comprises in situ amplifying the respectivestrain-specific barcode sequence 140 or in situ sequencing therespective strain-specific barcode sequence 140 from the geneticmaterial 100 by rolling circle amplification following excision,preferably in situ or in vivo excision, the respective strain-specificbarcode sequence 140 from the genetic material 100.

The respective strain-specific barcode sequence 140 is preferablyexcised from the genetic material 100 using digestion or transposeactivity. The method preferably also comprises making ssDNA from theexcised strain-specific barcode sequence 140 in an exonuclease reactionand circulation by hybridizing a padlock probe, or by ssDNA ligation orby dsDNA ligation following self-hybridization.

The at least one library-common primer-binding sequence 160, 170 canalternatively, or in addition, be used to sequence the respectivestrain-specific barcode sequence 140 from the genome 100. In such acase, the library-common primer-binding sequence 160 will correspond tothe anchor primer-binding sequence for in situ sequencing by ligation orprimer-binding sequence for in situ sequencing by synthesis.

The in situ sequencing may be performed directly on the genome 100 ofthe cell strains as described above. However, it is generally preferredto generate cDNA sequences 300 by reverse transcribing RNA sequences 200obtained by transcribing the construct in the genome 100 comprising thestrain-specific barcode sequence 140, the variable region 150 and the atleast one library-common primer-binding sequence 160, 170. In such acase, it is possible to in situ amplify the cDNA sequences 300 prior tostarting the in situ sequencing of the strain-specific barcode sequences340.

It is also possible to amplify the genomic region including the barcodesequence 140 directly by using an isothermal amplification technique,such as loop-mediated isothermal amplification (LAMP), stranddisplacement amplification (SDA), helicase-dependent amplification (HAD)or nicking enzyme amplification reaction (NEAR).

In another embodiment, a RCA template is formed by letting the cDNA 300self-ligate by using reverse transcription primers that mediatehybridization of the two ends and consecutive ligation. For instance,the cDNA 300 is ligated after it has self-hybridized to a nicked doublestranded DNA over, preferably, at least 6 basepairs.

In an embodiment the method comprises synthesizing, for each cellstrain, a cDNA sequence 300 from a transcribed RNA sequence 200 usingprimers 360 complementary to at least one library-common primer-bindingsequence 260 in the transcribed RNA sequence 200 and a reversetranscriptase or an RNA-dependent DNA polymerase. In such a case, the insitu sequencing preferably comprises in situ sequencing at least a partof the respective strain-specific barcode sequence 340 in the cDNAsequence 300 of each cell strain at the spatially defined and separatedpositions in the culturing device 1.

The in situ sequencing can be performed by in situ sequencing byligation or by synthesis of at least a part of the respectivestrain-specific barcode sequence 340.

The in situ sequencing of the strain-specific barcode sequences 340 canbe performed using the previously mentioned at least one library-commonprimer-binding sequence 360, 370. Alternatively, a dedicated sequencingprimer-binding sequence 130 could be provided in the genome 100,preferably directly upstream of the barcode sequence 140. Thissequencing primer-binding sequence 130 preferably has known nucleotidesequence and is advantageously library-common for all cell strains inthe library.

In this approach, the at least one library-common primer-bindingsequence 160, 170 is mainly used for reverse transcription andamplification purposes, whereas the sequencing primer-binding sequence130 is used for in situ sequencings of the strain-specific barcodesequences 140.

In a specific implementation only the barcode sequence 140 istranscribed along with flanking sequences 160, 170 for amplification orcircularization of the corresponding cDNA, see FIG. 11. The transcribedbarcode sequence 140 is integrated at a distance from the variableregion 150, but the mapping between the variable region 150 and thebarcode sequence 140 can be obtain through individual sequencing readswhen the corresponding region of the cell library is sequenced in bulk.The advantage of this implementation is that the barcode sequence 140and its flanking regions 160, 170 can be selected independent of theorganization of the variable region 150 as long as the barcode sequence140 has a much greater diversity than the variable region 150. Forexample a 15 base pair (bp) random barcode sequence will allow 4¹⁵˜10⁹different barcode combinations. If the corresponding variable region has10⁶ variants, the risk is only 0.001 that one barcode sequence match twodifferent variable regions.

FIG. 12 illustrates a cDNA sequence 300 obtained from the transcribedportion of the genome 100 shown in FIG. 11. The two library-commonprimer-binding sequences 360, 370 flanking the barcode sequence orregion 340 in the cDNA sequence 300 can be used for amplification,padlock probing or anchor-primer ligation as previously describedherein.

Once the nucleotide sequence of the strain-specific barcode sequence 140has been determined the mapping information can be used to get thecorresponding nucleotide sequence of the variable region 150 thatmatches this particular strain-specific barcode sequence 140. This ispreferably performed for each cell strain in the library to determinethe different genotypes. The result is thereby a respective genotype foreach spatially defined and separated position in the culturing device.The genotypes are then matched or connected to the previously determinedphenotypic characteristics to thereby provide information of theparticular genotypes that resulted in the determined phenotypiccharacteristics in the library of cell strains.

In an embodiment, the library of cell strains is characterized by thatthe different cell strains expresses different RNA products fromdifferentially barcoded extra chromosomal genetic elements. The RNAproducts could, for instance, be mRNA, iRNA, or guide RNA for dCas9.

Another aspect of the embodiments relates to a system for characterizinga library of a plurality of cell strains having different variableregions in at least one part of the genome of the cell strains. Thesystem comprises a culturing device configured to culture cells of thecell strains at spatially defined and separated positions in theculturing device. The system also comprises a first kit comprisingcomponents for in situ genotyping the variable region of each cellstrain following fixation of the cells at the spatially defined andseparated positions in the culturing device. With such a system, arespective phenotypic characteristic of each cell strain can beconnected to each respective genotype based on the spatially defined andseparated positions in the culturing device.

In an embodiment, the system also comprises a microscope configured todetermine a phenotypic characteristic and a genotype of each cell strainin the culturing device. Thus, a microscope of the system is used tomonitor and determine the respective phenotypic characteristics of thecell strains at the respective spatially defined and separated positionsin the culturing device. The microscope is preferably also used duringin situ sequencing in order to read out fluorescent signals.

In an embodiment, the system also comprises the library of the pluralityof cell strains. This library of cell strains can be generated aspreviously described herein using techniques for genome engineering togenerate a large library of different genotypes.

In such a library the mapping between the variable region and thebarcode sequence will be provided, such that preferably only the barcodesequence needs to be sequenced in order to determine the genotype.

The library can for example be based on conditional repression oractivation of the activity for each gene in cells from a specificorganisms. For example the variable region can encode one or a few shortguide RNAs for each transcribed region in the organism, such that thegene activity of individual genes can be altered by a dCas9 mediatedregulation.

The culturing device of the system can be constructed according tovarious embodiments. In an embodiment, the culturing device is aculturing device or plate comprising a plurality of wells, patches orcompartments, preferably at least one well, patch or compartment percell strain of the library. For instance, cell plates having severalthousands of wells, such as 96×96 wells, are available on the marked forcell culturing purposes. Such cell plates can be used as culturingdevice in the embodiments. In such a case, each well corresponds to aspatially defined and separated position in the culturing device. Theculture plate is preferably of plastic material or glass that istransparent for imaging or is built directly on a spatially addressablebiosensor or light detector array.

In another embodiment, the culturing device is a substrate having aplurality of spatially defined and separated patches where cells of thecell strains can adhere and grow. The substrate is preferably made of aplastic material or glass that is transparent for imaging. The substratemay, in an embodiment, be in the form of a microscope slide having aplurality of micro-wells constituting the spatially defined andseparated positions in the culturing device. The surface of themicro-wells may optionally be treated or coated for facilitating celladherence.

An alternative approach is to have a microscope slide or other substratehaving a plurality of patches that constitute respective portions of themicroscope slide or other substrate that have been surface treated orcoated to facilitate and promote cell adherence. This means that cellsof the library will easily adhere to and grow on the patches whereas thecells do not efficiently adhere to intermediate surface portions of themicroscope slide or other substrate lacking the adherence-promotingsurface treatment or coating. This means that by adding culture media tothe microscope slide or other substrate any cells present on theintermediate surface portions will be flushed away, whereas cellsgrowing on the patches remain firmly attached to the surface. Whenseeded sparsely this format will support the growth of isogenic cells ateach patch.

Patches or wells can be coated with for example poly-lysine, collagen,fibronectin, lamninin and/or gelatin.

More information of culturing devices that can be used according to theembodiments can be found in Wang et al. Current Biology 20, 1099-1103,2010.

In an embodiment, the culturing device is a microfluidic device 1, seeFIGS. 1-7. The microfluidic device 1 comprises a substrate 10transparent for imaging and having a plurality of spatially defined andseparated cell channels 20. The cell channels 20 have a dimension toaccommodate cells in monolayer. A respective end 22 of the plurality ofspatially defined and separated cell channels 20 is in fluid connectionwith a flow channel 30 having a fluid source 31 in a first end 32 of theflow channel 30 and a fluid sink 33 at a second end 34 of the flowchannel 30.

The substrate 10 has multiple cell channels 20 in which cells of thecell strains are cultured. The cell channels 20 may be arranged inparallel as shown in FIGS. 1 and 3-7 with a respective end 22 in fluidconnection with the flow channel 30 and extending from this flow channel30. In order to increase the total number of cell channels 20, the cellchannels 20 may extend from either longitudinal side of the flow channel30 thereby substantially doubling the number of cell channels 20 ascompared to only having cell channels 20 on one side of the flow channel30, see FIG. 6.

Also more complex arrangements of cell channels 20 and flow channels 30are possible in the substrate to increase the number of flow channels30, see FIG. 7. The important characteristic is that each cell channel20 has an end 22 in fluid connection with a flow channel 30 and that thecell channels 20 are separated to prevent cells from escaping from onecell channel 20 and entering another cell channel 20.

The cell channels 20 are dimensioned to accommodate cells in monolayer.This means that the height or diameter of the cell channels 20 isselected to be about or slightly larger than the diameter of the cellsin the library. For instance, the cell channels 20 could besubstantially quadratic in cross-section as shown in FIGS. 2 and 4 witha channel side substantially matching the cell diameter of the cells.Alternatively, the cell channels could have circular or U-shapedcross-section with a diameter substantially matching the cell diameter.Also other cross-sectional configurations are possible as long as thecells could be viably cultured, preferably in monolayer, in the cellchannels 20. This implies that the cell channels 20 can be several cellswide but preferentially only one cell high. In this case the cells cangrow in the cell channels forming a 2D monolayer that is may be widerthan one cell but preferably still is a monolayer. A 2D monolayergenerally facilitates phase contrast imaging of the cells as compared togrowing the cells in a single line, one cell wide.

The flow channel 30 preferably has dimensions that are significantlylarger than the diameter of the cells. This means that any cellsentering the flow channel 30 will be flushed through the flow channel 30towards the fluid sink 33 by a, preferably continuous, flow of culturemedium from the fluid source 31 through the flow channel 30 and towardsthe flow sink 33.

Cells of the library are seeded by adding cells of a respective cellstrain in each cell channel 20. The cells are thereby allowed to grow ina monolayer along the length of the cell channels 20. Each cell channelthereby contains cells of a single cell strain and genotype. The cellsin the cell channels 20 could be seen as pearls on a string if the cellchannel 20 is one cell wide. If the cell channel 20 is wider the cellswill form a 2D layer in the cell channel 20.

Cells growing and mitigating past the end 22 of the cell channels 20will enter the flow channel 30 and are thereby flushed away. The other,opposite end 28 of the cell channels 20 is preferably closed ordimensioned to prevent cells from escaping from this end 28.Alternatively, these ends 28 of the cell channels 20 could be in fluidconnection with a second flow channel. In such a case, any cellsescaping from the cell channels 20 will be flushed away from the flow ofculture medium in this second flow channel.

The fluid source 31 is used to input culture medium into the flowchannel 30 and further into the cell channels 20. The culture medium isallowed to exit through the flow sink 33. There is preferably acontinuous flow of culture medium from the fluid sink 31 through theflow channel 30 and into the cell channels 20 and out of the fluid sink33.

The culture medium preferably contains ingredients required by the cellsin the library to support viability and optionally also cell growth. Theparticular culture medium to use depends on the library of cell typesand can be selected by the user of the system.

It is also possible to add any chemical agent or drug as test agent tothe cells in the cell channels 20 using the fluid source 31, forinstance by adding at least one test agent to the culture medium thatenters the fluid source 31. In such a case, a phenotypic response of thecell strains to the at least one test agent in the culturing device 1can be determined, for instance, by microscopy.

The inner surfaces of the cell channels 20 or at least the bottomsurface thereof may be surface treated or coated to promote celladhesion and/or to reduce binding of enzymes and probes in enzymaticsteps.

FIG. 1 and FIG. 2, showing a cross-sectional view of the microfluidicdevice 1 in FIG. 1 along the line A-A, illustrate an embodiment that inrelation to state-of-the-art significantly improves flow of culturemedium through the cell channels 20 but also promotes cell loading,washing and incubation steps of the cells in the cell channels 20 prior,during and following fixation of the cells in the cell channels 20.

In the illustrated embodiment, each cell channel 20 is flanked along atleast one of its longitudinal sides 24, 26 with a respective washchannel 40 having a first end 42 in fluid connection with the flowchannel 30 and a second, opposite end 44 in fluid connection with a washsink 50. The wash channels 40 have a dimension that is too small toaccommodate cells.

In an embodiment, each cell channel 20 has at least one wash channel 40in fluid connection with the cell channel 20 and arranged along one ofits longitudinal sides 24, 26. The embodiment as shown FIGS. 1 and 2have wash channels 40 arranged along both longitudinal sides 24, 26 ofeach cell channel 20.

In an embodiment, the wash channels 40 may be interconnected, such as atone or both of their ends 44 thereby forming a continuous wash layeraround the cell channels 20.

The dimension of the wash channel 40 (or wash layer), such as depth,height and/or width, is too small to accommodate cells. This means thatcells present in the cell channels 20 cannot enter the adjacent washchannels 40 but will remain in the cell channels 20. FIG. 2 clearlyillustrates the comparatively smaller depth of the wash channels 40 ascompared to the cell channels 20. The wash channels 40 may have anycross-sectional configuration, such as quadratic, rectangular, circular,U-shaped, etc.

In an embodiment, the wash channels 40 form a wash layer as shown inFIG. 1. This means that wash channels 40 are present not only along thelongitudinal sides 24, 26 of the cell channels 20 but also extend fromthe respective second ends 28 of the cell channels 20 to the sinkchannel 52. Thus, a first set of wash channels 40 run along the cellchannels 20 and extend from the flow channel 30 to the sink channel 52.A second set of wash channels 40 starts from the second ends 28 of thecell channels 20 and ends at the sink channel 52. The first and secondsets of wash channels 40 together form a wash layer.

The cell channel 20 preferably has a substantially same depth whentraveling from its first end 22 at the flow channel 30 to its second end28. This depth preferably corresponds to or is slightly larger than thecell diameter to allow cells in a monolayer in the cell channel 20. Atthe second end 28 of the cell channel 40 the depth will be shallowerwhen entering the wash channel 40 extending from the second 28 to thesink channel 52. This shallower depth, which preferably is smaller thanthe cell diameter, prevents cells present in the cell channel 20 fromentering the wash channel 40.

Herein follows a short description of the operation of the microfluidicdevice 1.

During cell loading, the cells with media enter the fluid source 31 andflow into the flow channel 30. In a preferred embodiment, both the fluidsink 33 and the wash sink 40 are open to allow media and cells to bepushed into the cell channels 20. Excess cells are washed out throughthe fluid sink 33 as the depth of the wash channels 40 (wash layer) istoo shallow to allow the cells entering the sink channel 52 and reachthe wash sink 50. Media exits the microfluidic device 1 both from thewash sink 50 and the fluid sink 33.

During operation of the microfluidic device 1 culture medium enters thefluid source 31 as described above. In a first embodiment, the wash sink50 and the fluid sink 33 are open. This means that culture medium willnot only exit through the fluid sink 33 but also through the wash sink50. This means that the culture medium and reagents for in situsequencing will effectively reach all cells within the cell channels 20.Excess cell will flow into the flow channel 30 and further out from thefluid sink 33, whereas media flow over all the cells and into the sinkchannel 50 and out from the wash sink 50 to keep all cells supplied withfresh culture medium.

In a second embodiment, the wash sink 50 is closed so that culturemedium and excess cells both exit through the fluid sink 33. This isembodiment generally achieves a less efficient flow of cell medium overthe cells in the cell channels as compared to the first embodiments.

In wash and reaction steps, the wash sink 50 is preferably open. Thismeans that the wash fluid or liquid or the solution with reactionreagents enters the fluid source 31 and flows through the flow channel30, the cell channels 20 and the wash channels 40 towards the wash sink50. In an embodiment, the fluid sink 33 is closed during washing steps.In another embodiment, the fluid sink 33 is open during washing steps.In such a case, wash fluid or liquid may exit through the wash sink 50or the fluid sink 33.

Washing of cells may take place prior to fixation of the cells in thecell channels 20 to wash away any culture medium, during the fixation ofthe cells and/or following fixation of the cells to wash away thefixation chemicals, such as formaldehyde. Washing and reaction steps mayalso be performed in connection with the in situ genotyping when thereis a need to change reaction components and reaction medium.

The wash channels 40 are preferably interconnected in their ends 44 by asink channel 52 that is in fluid connection with the wash sink 50. Inthe figures, this sink channel 52 goes parallel with the flow channelwith the wash channels 40 extending there between. However, whereas thecell channels 20 have one end 22 in fluid connection with the flowchannel 30 the other end preferably ends a distance from the sinkchannel 52 to prevent cells from exiting the cell channels 20 into thesink channel 52. If the wash sink 50 is open during culturing of thecells so that there is a flow of culture medium from the fluid source 31towards not only the fluid sink 33 but also towards the wash sink 50this enables uniform growth conditions throughout the cell channel 20.

The cell channels 20 and the wash channels 40 are preferably openchannels as shown in FIG. 2. This means that a cover plate 70 ispreferably positioned on the substrate 10 to form a lid for and seal thecell channels 20 and the wash channels 40.

The substrate 10 preferably comprises structures or portions 60extending through the whole thickness of the substrate 10 in order toincrease its stability. FIGS. 1 and 2 illustrate such structures 60 inthe form of pillars provided in between some of the wash channels 40along the longitudinal lengths of the cell channels 20. These pillarscan have any shape as long as they support the wash layer and provideflow. They could, for example, be rectangular, star shaped, round ortriangular and positioned regularly or irregularly. These structures 60could be separate structures as shown in the figures to promote flow ofwash liquid throughout the whole wash layer, i.e. in between washchannels 40. In an alternative approach, each column of pillars shown inthe figures forms a single structure extending over the whole lengthbetween the flow channel 30 and the sink channel 52. Such a solution mayresult in a more stable substrate 10, however, at the cost of lessefficient washing.

FIG. 5 illustrates another embodiment of the microfluidic culture device1. This embodiment lacks the wash channels running along thelongitudinal sides 24, 26 of the cell channels 20. In clear contrast,supporting structures 60 are present between adjacent cell channels 20and extend from the flow channel 30 to the sink channel 52 as shown inthe figure. Wash channels 40 are preferably present and extend from thesecond ends 28 of the cell channels 20 to the sink channel 52. Hence, awash channel 40 has a first end 42 in fluid connection with a second end28 of a cell channel 20 and a second, opposite end 44 in fluidconnection with the sink channel 52. This embodiment generally providesa more stable microfluidic device 1 as compared to using pillar-likestructures as shown in FIG. 1.

The structure of cell channels 20 can be multiplexed, see for exampleFIGS. 6 and 7. The common feature being that cells are mechanicallyconstricted to grow in a monolayer and that they are flooded with media,buffers, enzymes etc. without being washed away since they arephysically too big to be pushed into the wash channels 40. It alsorequires two sinks for the cell channels, one to accommodate the excesscells that do not fit in a monolayer, i.e. the fluid sink 33, and one toaccommodate the media that flows over the cells, i.e. the wash sink 50.

The microfluidic device 1 of FIG. 6 basically multiplexes two structuresas shown in FIG. 5. This means that the two structures of cell channels20 and wash channels 40 (left and right in the figure) share a commonflow channel 30 connected in its first end 32 to the fluid source 31 andin its second end 34 to the fluid sink 33. Each structure of cellchannels 20 and wash channels 20 ends at a respective sink channel 52that are interconnected and connected to a common wash sink 50.

The cells present in cell channels 20 in the structure to the left willbe exposed to the same culture medium and reagents and chemicals inputat the fluid source 31 as the cells present in the cell channels in thestructure to the right in the figure.

FIG. 7 illustrates an embodiment of a microfluidic device 1 havingmultiple, i.e. at least two, structures of cell channels 20 and washchannels 40 share a common fluid sink 33 and wash sink 50 but haveseparate, i.e. individual, fluid sources 31. This means that differentculture medium and/or reagents or chemicals can be input to cellspresent in one of the structures of cells channels 20 as compared tocells present in another of the structures of cells channels 20.

During loading, cells and culture medium enters the fluid sink 33 withmedia flowing out through the wash sink 50 and the separate fluidsources 31, whereas excess cells flow out through the fluid sources 31.In another embodiment, the cells enter the individual fluid sources 31with excess cells exiting the common fluid sink 33. During operation,media enter the separate fluid sources 31, thereby allowing differentmedia to enter the different structures of cells channels 20 and washchannels 40. Excess cells are washed out through the fluid sink 33 andmedia flow out from the wash sink 50 and also out through the commonfluid sink 33.

FIGS. 3 and 4 illustrate another embodiment of the microfluidic device 1that lacks wash channels, wash source and wash sink. In clear contrast,the microfluidic device 1 comprises a semipermeable membrane 80 havingan average pore size that is smaller than an average diameter of thecells. The semipermeable membrane 80 is arranged on the substrate 10 toform a lid for the plurality of spatially defined and separated cellchannels 20.

In this embodiment, the cell channels 20 are open channels having anopening in one of the main surfaces 12 of the substrate 10. Thesemipermeable barrier 80 is then positioned on this main surface 12 toform a lid for the cell channels 20.

During washing, the washing fluid or liquid may efficiently flow throughthe cell channels 20 from the fluid source 31 and flow channel 30 andout through semipermeable barrier 80. The average pore size of thesemipermeable barrier 80 is selected to prevent the cells from passingthrough the semipermeable barrier 80 but allow the washing fluid orliquid to pass there through.

The substrate 10 may be made in any transparent material, such asplastic material, in which the structures constituting the cell channels20, the fluid source 31, the fluid sink 33, the flow channel 30 andoptionally the wash channels 40, sink channel 52 and wash sink 50 can bedefined. Non-limiting examples of suitable materials include ZEONEX® andZEONOR®, which are cyclic olefin polymers (COP) marketed by ZEONChemicals L.P. and TOPAS®, which are cyclic olefin copolymers (COC)marketed by Topas Advanced Polymers. These materials have excellentoptical characteristics in terms of transmission and backgroundfluorescence. They also have good flow characteristics when heated andmay therefore replicate small structures allowing formation ofsubstrates 10 as shown in FIGS. 1-7.

Other examples of suitable materials for the substrate 10 includeglasses, polydimethylsiloxane (PDMS), poly(methyl methacrylate) (PMMA),polycarbonate (PC), polypropylene (PP), polytetrafluoroethylene (PTFE),polyethylene terephthalate (PET) and poly(p-phenylene sulfide) (PPS).

The semipermeable membrane 80 can be selected among dialysis membranes,such as marketed by Thermo Fisher Scientific Inc. Alternatively, thesemipermeable membrane 80 may be manufactured from any of the plasticmaterials mentioned above for the substrate 10.

The cover plate 70 may be manufactured in various materials that arepreferably transparent to allow imaging. Non-limiting examples includeglass and plastic materials.

In an embodiment, the system also comprises a fluidic manifoldconfigured to distribute components of the first kit to the cellchannels 20 using at least one computer-controlled pump. The fluidicmanifold is preferably configured to enable change of media forphenotyping, distribution of chemicals used for cell fixation and applyneeded for in situ sequencing using the computer-controlled andpreprogramed pumps. In a particular embodiment, the reagents and cellculture media can be maintained at different temperatures throughout theexperiment.

In an embodiment, each cell strain has a respective strain-specificbarcode sequence in its genome or a mobile genetic element. The firstkit then comprises components for determining the respective genotype byin situ sequencing at least a part of the respective specific barcodesequence of each cell strain at the spatially defined and separatedpositions in the culturing device.

The system of the embodiments may, for instance, be used for phenotypingand genotyping bacteria. In such a case, the cell channels 20 preferablyhave dimensions between 800-1200 nm and the wash channels 40 arepreferably less than 400 nm high. In a particular embodiment, themicrofluidic device 1 preferably comprises more than 1000 cell channels20 that are advantageously individually marked and, thus, recognizable.

The microfluidic device 1 of the embodiments is highly suitable forusage in the system for characterizing a library of a plurality of cellstrains. However, the microfluidic device 1 could alternatively be usedfor other purposes than allowing determination of both phenotype andgenotype of a library of cell strains, such as screening for antibioticresistance and other screening operations.

Hence, an aspect of the embodiments relates to a microfluidic device 1comprising a substrate 10 transparent for imaging and having a pluralityof spatially defined and separated cell channels having a dimension toaccommodate cells in monolayer. A respective first end 22 of theplurality of spatially defined and separated cell channels 20 is influid connection with a flow channel 30 having a first end 32 in fluidconnection with a fluid source 31 and a second end 34 in fluidconnection with a fluid sink 33. A respective second end 28 of theplurality of spatially defined and separated cell channels 20 is influid connection with a first end 42 of a respective wash channel 40having a second end 44 in fluid connection with a sink channel 52, whichis in fluid connection with a wash sink 50. The wash channels 40 have adimension too small to accommodate cells.

In an embodiment, each cell channel 20 is flanked along at least one ofits longitudinal sides 24, 26 with a respective second wash channel 40having a first end 42 in fluid connection with the flow channel 30 and asecond end 44 in fluid connection with the wash channel 52.

In an embodiment, the first kit comprises components for in situsequencing by ligation of the at least a part of the respective specificbarcode sequence at the spatially defined and separated positions in theculturing device.

In this embodiment, the first kit preferably comprises a DNA ligase, ananchor primer having a nucleotide sequence that is complementary to thenucleotide sequence of a library-common primer-binding sequence in thegenome of the cell strains, a mixed pool of labelled interrogation probeoligonucleotides. The first kit preferably also comprises a reactionmixture containing components required for the DNA ligase (ATP, buffer)to join a labelled probe oligonucleotide to the anchor primerbase-pairing with the library-common primer-binding sequence and forcleaving the hybridized probe at uracil (uracil-DNA glycosylase).

In another embodiment, the first kit comprises components for in situsequencing by synthesis of the at least a part of the respectivespecific barcode sequence at the spatially defined and separatedpositions in the culturing device.

In this embodiment, the first kit preferably comprises four types ofmodified dNTPs containing a reversible terminator that contains afluorescent label. The first kit preferably also comprises a DNApolymerase and a sequencing primer having a nucleotide sequence that iscomplementary to the nucleotide sequence of a library-commonprimer-binding sequence in the genome of the cell strains. The first kitalso comprises a reaction mixture containing components required for theDNA polymerase to incorporate the modified nucleotides to the sequencingprimer.

The system preferably also comprises mapping information specifying aconnection between each variable region and a respective strain-specificbarcode sequence. The respective genotype is then determined based onthe in situ sequenced at least a part of the respective strain-specificbarcode sequence and the mapping information.

In an embodiment, each cell strain of the library has a respectiveconstruct comprising a respective strain-specific barcode sequence andthe variable region in its genome flanked by library-commonprimer-binding sequences of known nucleotide sequence. Thelibrary-common primer-binding sequences can thereby be used to amplifythe respective strain-specific barcode sequence from the genome or via atranscribed RNA sequence that is reverse transcribed into a cDNAsequence.

In an embodiment, the system comprises a second kit comprisingcomponents for synthesizing, for each cell strain, a cDNA sequence froma transcribed RNA sequence using primers complementary to at least onelibrary-common primer-binding sequence in the transcribed RNA sequenceand a reverse transcriptase or an RNA-dependent DNA polymerase. Thefirst kit then preferably comprises components for in situ sequencingthe at least part of the respective strain-specific barcode sequence inthe cDNA sequence of each cell strain at the spatially defined andseparated positions in the culturing device.

The second kit preferably comprises a primer sequence that iscomplementary to at least one library-common primer-binding sequence inthe transcribed RNA sequence and the reverse transcriptase orRNA-dependent DNA polymerase. The second kit also comprises thenucleotides that are used by the enzyme to generate the cDNA sequence.The second kit also comprises a reaction mixture containing componentsrequired for the reverse transcriptase or RNA-dependent DNA polymeraseto incorporate the nucleotides, such as the reverse transcriptase,dNTPs, RNAse inhibitors. The second kit preferably also comprises anRNase to degrade residual RNA following the reverse transcription.

In an embodiment, the system also comprises a third kit comprisingcomponents (DNA polymerases, ligase, dNTPs) for in situ amplifying thecDNA sequence by rolling circle amplification following circulationusing ssDNA ligase or a padlock probe, or by in situ PCR.

In an embodiment, the system further comprises a fourth kit comprisingcomponents for fixating the cells at the spatially defined and separatedpositions in the culturing device. This fourth kit preferably comprisesformaldehyde that can be used to fixate the cells.

The components of the kits in the system can be provided as separatecomponents in dedicated containers. Alternatively, at least some of thecomponents of a kit may be provided a as a mixture present in a samecontainer.

The method and system of the embodiments can be used in variousapplications in where there is a need to phenotypically characterize alibrary of cell strains and associating the various phenotypes to thedifferent genotypes in the library.

For instance, the embodiments can be used to optimize fluorescentproteins or RNA aptamers to select for fast maturing intracellularsignals. In this application the protein or RNA-coding region of thereporter, i.e. a fluorescent protein or the apatmer, would be encoded inthe variable region of the genome. The expression of the reporter may beinduced by a chemical agent, such as isopropylβ-D-1-thiogalactopyranoside (IPTG) of the reporter is regulated the bythe lac repressor protein. The culture media or cells also contain anycofactor that is needed for the reporter florescence. The phenotype ismonitored by measuring fluorescence over time after induction and thephenotypes are scored by the time for reaching a specific level offluorescence or the fluorescence that is reached after a specific timeor the time constant for relaxation to the steady-state fluorescencevalue.

In an embodiment, the library of cell strains could be a library inwhich genes can be downregulated by a chemical signal, such as theexpression of a guide RNA for dCAS9. The cell strains could then bebarcoded with known mapping information between the barcode and theparticular gene.

Furthermore, the embodiments can be used to identify gene regulatory RNAsequences or proteins by sensitive detection of regulator properties invivo. Thus, the variable regions then encode different such generegulatory RNA sequences or proteins. The regulatory effect of the RNAsequences or proteins can then be monitored, for instance by microscopy,in order to detect various phenotypic characteristics that are caused bythe gene regulation.

The embodiments may also be used to select proteins or peptides forinhibition or activation of biological processes in the cells. Thevariable regions then encode different versions of the proteins orpeptides. The phenotypic monitoring then involves monitoring for theinhibition or activation of the relevant biological process.

Furthermore, the variable regions in the cell strains of the library maybe a set of expressed sequences that repress or enhance expression orregulation of specific genes. This can for example be interference RNA(iRNA) or short guied mRNA (sgRNA) sequences that can be constitutivelyexpressed or conditional. The phenotypes can be screened for differencesin phenotype, such as problems in development, growth, differentiation,and in their different responses to added test agents.

The embodiments can further be used to identify which genes that arerequired in specific steps in differentiation cells, such as stem cells,or in responses to a test agent. The variable regions may encodedifferent sgRNA or iRNA that repress expression of different genes, suchthat different gene are effectively shut off in different cells. Thephenotypic characterization may be in the form of monitoring thedifferentiation of cells in response to chemical test agents, such asgrowth factors or a drug. If cells, tissues or organisms display analteration in some steps of differentiation, the gene that has alteredexpression in that cell strain is related to the alerteddifferentiation. Using a very similar approach, it is possible to studyand determine the genes that are important for development inmulticellular organisms or tissues or proliferation of cancer cells.

It is also possible to screen for regulatory sequences that respond to aspecific stimuli by monitoring reporter protein expression with amultitude of regulatory sequences. Hence, the variable regions thencorrespond to the regulatory sequences. The phenotypic determination isthen based on monitoring reporter protein expression in the cells andwhere the gene encoding the reporter gene is under regulatory control ofthe regulatory sequences. The cells are then exposed to specificstimuli, such as physical stimuli, e.g. temperature changes, or chemicalstimuli, e.g. addition of test agent, and the response thereto ismonitored through the reporter gene expression.

The method and system of the embodiments can be used to identifyinteraction partners and regulators of a gene or gene product bymonitoring the localization, diffusion or concentration of the gene orgene product in a library where other gene products are selectively andconditionally knocked down. In this case, the variable regions mayencode different products that achieve the selective and conditionalknock down, for instance iRNA and sgRNA. In a similar application, onecan use a knock down library in combination with phenotypic monitoringof a fluorescently tagged protein of interest in order to study itsintracellular diffusion, localization or concentration. The libraryscreen could in this case directly identify potential interactionpartners or regulators.

A barcoded genomic mutation library can be made in parallel byintroducing three elements to a cell, for example by having them in thesame plasmid. The three elements include (1) a homologues but partiallymutated DNA sequence corresponding to the desired introduced change(variable region), (2) a guide RNA for CRISPR-mediated DNA cleavage ofthe corresponding unmutated DNA sequences and (3) a barcode sequence forin situ sequencing. The barcode sequence can be maintained on a mobilegenetic element or be introduced at a separate position on thechromosome.

When it is possible to monitor the biosynthesis of a compound ofinterest in the microscope or by a biosensor integrated in the culturingdevice this can be used as phenotypic readout to optimize expressionlevels for enzymes in a biosynthesis pathway or their amino acidsequences.

The above described applications should merely be seen as a few butillustrative uses of the method and system of the embodiments.

The embodiments described above are to be understood as a fewillustrative examples of the present invention. It will be understood bythose skilled in the art that various modifications, combinations andchanges may be made to the embodiments without departing from the scopeof the present invention. In particular, different part solutions in thedifferent embodiments can be combined in other configurations, wheretechnically possible. The scope of the present invention is, however,defined by the appended claims.

1.-28. (canceled)
 29. A method of characterizing a library of aplurality of cell strains having different variable regions in at leastone part of the genetic material of said cell strains, said methodcomprises: culturing cells of said cell strains at spatially defined andseparated positions in a culturing device; determining a phenotypiccharacteristic of each cell strain in said culturing device; fixatingsaid cells of said cell strains at said spatially defined and separatedpositions in said culturing device; in situ genotyping said variableregion of each cell strain at said spatially defined and separatedpositions in said culturing device; and connecting each respectivephenotypic characteristic to each respective genotype based on saidspatially defined and separated positions in said culturing device. 30.The method according to claim 29, wherein in situ genotyping comprisesin situ sequencing by ligation of said variable region or at least aportion thereof at said spatially defined and separated positions insaid culturing device.
 31. The method according to claim 29, wherein insitu genotyping comprises in situ sequencing by synthesis of saidvariable region or at least a portion thereof at said spatially definedand separated positions in said culturing device.
 32. The methodaccording to claim 29, wherein each cell strain has a respectivestrain-specific barcode sequence in its genetic material and in situgenotyping comprises determining said respective genotype by in situsequencing at least a part of said respective strain-specific barcodesequence of each cell strain at said spatially defined and separatedpositions in said culturing device.
 33. The method according to claim32, further comprising: determining mapping information specifying aconnection between each variable region and a respective strain-specificbarcode sequence, wherein determining said respective genotype comprisesdetermining said respective genotype based on said in situ sequenced atleast a part of said respective strain-specific barcode sequence andsaid mapping information.
 34. The method according to claim 33, whereindetermining said mapping information comprises sequencing said libraryof cell strains in bulk to obtain, for each cell strain, a sequence readencompassing said variable region and said respective strain-specificbarcode sequence.
 35. The method according to claim 32, wherein eachcell strain has a respective construct comprising a respectivestrain-specific barcode sequence and said variable region in its geneticmaterial flanked by at least one library-common primer-binding sequenceof known nucleotide sequences that can be used to amplify saidrespective strain-specific barcode sequence or sequence said respectivestrain-specific barcode sequence from said genetic material or via atranscribed ribonucleic acid, RNA, sequence that is reverse transcribedinto a complementary deoxyribonucleic acid, cDNA, sequence.
 36. Themethod according to claim 35, further comprising synthesizing, for eachcell strain, a cDNA sequence from a transcribed RNA sequence usingprimers complementary to at least one library-common primer-bindingsequence in said transcribed RNA sequence and a reverse transcriptase oran RNA-dependent DNA polymerase, wherein in situ sequencing comprises insitu sequencing said at least part of said respective strain-specificbarcode sequence in said cDNA sequence of each cell strain at saidspatially defined and separated positions in said culturing device. 37.The method according to claim 35, further comprising in situ amplifyingsaid cDNA sequences by rolling circle amplification followingcirculation using single-stranded DNA, ssDNA, ligase or a padlock probeor by in situ polymerase chain reaction, PCR, or by ligating said cDNAsequences after self-hybridization.
 38. The method according to claim35, further comprising in situ amplifying said respectivestrain-specific barcode sequence or in situ sequence said respectivestrain-specific barcode sequence from said genetic material by rollingcircle amplification following excision of said respectivestrain-specific barcode sequence from said genetic material.
 39. Themethod according to claim 32, wherein in situ sequencing comprises insitu sequencing by ligation of said at least a part of said respectivestrain-specific barcode sequence at said spatially defined and separatedpositions in said culturing device.
 40. The method according to claim32, wherein in situ sequencing comprises in situ sequencing by synthesisof said at least a part of said respective strain-specific barcodesequence at said spatially defined and separated positions in saidculturing device.
 41. The method according to claim 29, whereindetermining said phenotypic characteristic comprises determining saidphenotypic characteristic of each cell strain during culturing of saidcells in said culturing device using microscopy.
 42. The methodaccording to claim 29, further comprising adding at least one test agentto said cells in said culturing device, wherein determining saidphenotypic characteristic comprises determining a phenotypic response ofeach cell strain to said at least one test agent in said culturingdevice.
 43. The method according to claim 29, further comprisingrandomly seeding cells of said cell strains at said spatially definedand separated positions in said culturing device so that each spatiallydefined and separated position only comprises cells of a same genotype.44. A system for characterizing a library of a plurality of cell strainshaving different variable regions in at least one part of the geneticmaterial of said cell strains, said system comprises: a culturing deviceconfigured to culture cells of said cell strains at spatially definedand separated positions in said culturing device; and a first kitcomprising components for in situ genotyping said variable region ofeach cell strain following fixation of said cells at said spatiallydefined and separated positions in said culturing device, wherein arespective phenotypic characteristic of each cell strain can beconnected to each respective genotype based on said spatially definedand separated positions in said culturing device.
 45. The systemaccording to claim 44, further comprising a microscope arranged toenable determination of a phenotypic characteristic and a genotype ofeach cell strain in said culturing device.
 46. The system according toclaim 44, further comprising said library of said plurality of cellstrains.
 47. The system according to claim 44, wherein said culturingdevice is a microfluidic device comprising a substrate transparent forimaging and having a plurality of spatially defined and separated cellchannels having a dimension to accommodate cells in monolayer, wherein arespective first end of said plurality of spatially defined andseparated cell channels is in fluid connection with a flow channelhaving a fluid source at a first end of said flow channel and a fluidsink at a second end of said flow channel and a respective second end ofsaid plurality of spatially defined and separated cell channels is influid connection with a first end of a respective wash channel having asecond end in fluid connection with a sink channel with a wash sink,wherein said wash channels have a dimension too small to accommodatecells.
 48. The system according to claim 47, wherein each cell channelis flanked along at least one of its longitudinal sides with arespective second wash channel having a first end in fluid connectionwith said flow channel and a second, opposite end in fluid connectionwith said sink channel, said second wash channels having a dimensionthat is too small to accommodate cells.
 49. The system according toclaim 47, further comprising a fluidic manifold configured to distributesaid components of said first kit to said cell channels using at leastone computer-controlled pump.
 50. The system according to claim 44,wherein each cell strain has a respective strain-specific barcodesequence in its genetic material and said first kit comprises componentsfor determining said respective genotype by in situ sequencing at leasta part of said respective strain-specific barcode sequence of each cellstrain at said spatially defined and separated positions in saidculturing device.
 51. The system according to claim 50, furthercomprising mapping information specifying a connection between eachvariable region and a respective strain-specific barcode sequence,wherein said respective genotype is determined based on said in situsequenced at least a part of said respective strain-specific barcodesequence and said mapping information.
 52. The system according to claim50, wherein each cell strain has a respective construct comprising arespective strain-specific barcode sequence and said variable region inits genetic material flanked by at least one library-common sequence ofknown nucleotide sequences that can be used to amplify said respectivestrain-specific barcode sequence or sequence said respectivestrain-specific barcode sequence from said genetic material or via atranscribed ribonucleic acid, RNA, sequence that is reverse transcribedinto a complementary deoxyribonucleic acid, cDNA, sequence.
 53. Thesystem according to claim 52, further comprising a second kit comprisingcomponents for synthesizing, for each cell strain, a cDNA sequence froma transcribed RNA sequence using primers complementary to at least onelibrary-common primer-binding sequence in said transcribed RNA sequenceand a reverse transcriptase or an RNA-dependent DNA polymerase, whereinsaid first kit comprises components for in situ sequencing said at leastpart of said respective strain-specific barcode sequence in said cDNAsequence of each cell strain at said spatially defined and separatedpositions in said culturing device.
 54. The system according to claim52, further comprising a third kit comprising components for in situamplifying said cDNA sequences by rolling circle amplification followingcirculation using single-stranded DNA, ssDNA, ligase or a padlock probeor by in situ polymerase chain reaction, PCR, or by ligating said cDNAsequence after self-hybridization.
 55. The system according to claim 50,wherein said first kit comprises components for in situ sequencing byligation of said at least a part of said respective strain-specificbarcode sequence at said spatially defined and separated positions insaid culturing device.
 56. The system according to claim 50, whereinsaid first kit comprises components for in situ sequencing by synthesisof said at least a part of said respective strain-specific barcodesequence at said spatially defined and separated positions in saidculturing device.